Journal Pre-proofs Identification of a subset of trace amine-associated receptors and ligands as po- tential modulators of insulin secretion Michael J. Cripps, Marta Bagnati, Tania A. Jones, Babatunji W. Ogunkolade, Sophie R. Sayers, Paul W. Caton, Katie Hanna, Merell Billacura, Kathryn Fair, Carl Nelson, Robert Lowe, Graham A. Hitman, Mark D. Berry, Mark D. Turner PII: S0006-2952(19)30384-3 DOI: https://doi.org/10.1016/j.bcp.2019.113685 Reference: BCP 113685 To appear in: Biochemical Pharmacology Received Date: 22 August 2019 Accepted Date: 24 October 2019 Please cite this article as: M.J. Cripps, M. Bagnati, T.A. Jones, B.W. Ogunkolade, S.R. Sayers, P.W. Caton, K. Hanna, M. Billacura, K. Fair, C. Nelson, R. Lowe, G.A. Hitman, M.D. Berry, M.D. Turner, Identification of a subset of trace amine-associated receptors and ligands as potential modulators of insulin secretion, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.113685 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved. Identification of a subset of trace amine-associated receptors and ligands as potential modulators of insulin secretion Michael J. Cripps1, Marta Bagnati2, Tania A. Jones2, Babatunji W. Ogunkolade2, Sophie R. Sayers3, Paul W. Caton3, Katie Hanna1, Merell Billacura1, Kathryn Fair1, Carl Nelson1, Robert Lowe2, Graham A. Hitman2, Mark D. Berry4, Mark D. Turner1 1Centre for Diabetes, Chronic Diseases and Ageing, School of Science and Technology, Nottingham Trent University, Clifton, Nottingham, NG11 8NS, UK. 2Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK. 3Diabetes and Nutritional Sciences Division, King's College London, London, United Kingdom, SE1 1UL, 4Department of Biochemistry, Memorial University of Newfoundland, St. John's, A1B 3X9, Canada. Running title: Trace amine associated receptors in insulin secretion *Correspondence to: Dr. Mark D. Turner, School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, United Kingdom. E-mail: [email protected] Phone: +44(0) 115 848-3113 1 ABSTRACT The worldwide prevalence of diabetes has reached 8.5% among adults, and this is characterised by elevated glucose concentrations and failing insulin secretion. Furthermore, most people with type 2 diabetes are either obese or overweight, with the associated dyslipidaemia contributing to the development of insulin resistance and increased cardiovascular risk. Here we incubated INS-1 pancreatic β-cells for 72h in RPMI-1640 media, or media supplemented with 28mM glucose, 200µM palmitic acid, and 200µM oleic acid as a cellular model of diabetic glucolipotoxicity. Illumina HiSeq gene expression analysis showed the trace amine-associated receptor (TAAR) family to be among the most highly downregulated by glucolipotoxicity. Importantly, MetaCore integrated knowledge database, from Clarivate Analytics, indicated potential TAAR impact on insulin secretion through adenylyl cyclase signalling pathways. We therefore investigated the effect of TAAR ligands on cAMP signalling and insulin secretion, and found that only the branch of the TAAR family tree that is activated by isopentylamine, 2-phenylethylamine, p-tyramine, and agmatine significantly increased intracellular cAMP and resulted in increased insulin secretion from INS-1 cells and primary mouse islets under normal conditions. Crucially however, this enhancement was not evident when the receptor family was downregulated by glucolipotoxic conditions. This data indicates that a subset of TAARs are regulators of insulin secretion in pancreatic β-cells, and that their downregulation contributes to glucolipotoxic inhibition of insulin secretion. As such they may be potential targets for treatment of type 2 diabetes. Keywords: G-protein coupled receptor, adenylyl cyclase, cAMP, glucotoxicity, lipotoxicity 2 1. INTRODUCTION Over 80% of patients with type 2 diabetes (T2D) are either overweight or obese, and typically this is associated with insulin resistance. Consequently, these patients have both elevated blood glucose and free fatty acid concentrations (1). It is known that chronic hyperglycaemia inhibits stimulated insulin secretion, but it is the combination of both high sugar and high fat that has the largest detrimental effect on -cell function (2). We previously employed microarray technology to investigate the effect of glucolipotoxicity upon INS-1 pancreatic - cell gene expression, and found a wide spectrum of damaging effects including diminished insulin secretion, chronic inflammation, increased apoptosis, biological oxidation, and dysregulated nucleic acid processing and repair (3). Furthermore, based on ranked statistical significance of enrichment following MetaCore integrated knowledge database analysis (https://clarivate.com/products/metacore) of that dataset, we also demonstrated the presence of disease association with both endocrine and metabolic disorders, which indicates the presence of pathways in INS-1 cells with known T2D and obesity aetiology in man (3). Although microarray technology has proven successful, the development of next generation sequencing (RNAseq) technology offers a number of advantages over microarray approaches. For example, microarray analysis is limited by the need to rely upon existing knowledge of genome sequence, they have high background levels due to cross-hybridization, and a limited dynamic range of detection due to both background and saturation of signals. By contrast, next generation sequencing strategies analyse total amount of reads that map to the exons of a gene, normalised by the length of the exons, and do not have an upper limit for quantification (4). The presence and amount of each RNA can therefore be calculated and subsequently compared with the amount in any other sequenced sample. In addition, RNAseq data also have high levels of reproducibility for both technical (repeated measurements of the same sample) and biological (parallel measurements of biologically distinct samples) replicates. 3 Building from our previous work (2,3), we utilised Illumina HiSeq gene expression analysis to more accurately define the pathophysiology of type 2 diabetes, with a view to identifying potential new targets for therapeutic intervention. Data presented here indicate that downregulation of trace amine-associated receptor signalling contributes to the failure of insulin secretion that results from chronic exposure of pancreatic -cells to high concentrations of glucose and fatty acids. Furthermore, under control conditions, only stimulation of family members associated with one specific arm of the TAAR phylogenic tree enhanced insulin secretion. We propose that future studies focus on this specific subset of family members as potential new therapeutic targets for treatment of type 2 diabetes. 2. MATERIALS AND METHODS 2.1 Materials Antibodies were obtained from Abcam (Cambridge, UK) and Agilent Technologies (Santa Clara, CA, USA). Unless otherwise stated, all other chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) or VWR International Ltd (Lutterworth, UK). 2.2 Islet Isolation and INS-1 β-Cell Culture Islets were isolated from male CD1 mice by collagenase injection into the pancreatic duct. Digested pancreas was washed with MEM-2279 and separated from exocrine tissues by centrifuging through a Histopaque 1.077 g/ml gradient. After washing, islets were picked and incubated at 37 °C in RPMI-1640 (supplemented with 10% [vol/vol] fetal calf serum, 2 mM glutamine and 100U/ml penicillin/ 0.1 mg/ml streptomycin) for 24h prior to further analysis. INS-1 rat pancreatic -cells were cultured in RPMI-1640 media, or RPMI media 4 supplemented with 28mM glucose, 200µM oleic acid, and 200µM palmitic acid (GLT media), for 72h as detailed previously (2). All animal procedures were approved by the King’s College London Ethics Committee and carried out in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986. 2.3 Cell Viability INS-1 cells were cultured in either RPMI 1640 or GLT media for 72h. Media was aspirated and cells washed three times in modified Krebs-Ringer solution (KRB) (125mM NaCl, 1.2mM KH2PO4, 5 mM KCl, 2mM Mg SO4, 1mM CaCl2, 1.67mM glucose, 0.1% BSA, 25mM HEPES, pH7.4). Cells were then incubated in KRB media supplemented with a final concentration of 5µM Calcein AM Cell Viability Dye (ThermoFisher) for 1h, before being washed in KRB for a final time. Cell viability was measured via fluorescence, with excitation and emission at 490nm and 520nm respectively. 2.4 RNA Isolation Cells were incubated in the appropriate conditions for 72h, harvested and RNA extracted using an RNA isolation kit (Life Technologies, UK). RNA integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA), with all samples analysed in this study having a RIN score >8. Samples for transcriptome analysis were prepared using a Truseq™ RNA Sample Prep